This invention relates to the field of medical devices. In particular, the invention relates to a system for rendering a delivery vehicle suitable for delivery of an agent to a vertebrate, and a method of using the same.
The delivery of therapeutic agents to specific tissues is desirable to ensure that a sufficiently high dose of a given agent is delivered to a selected tissue while avoiding undesirable side effects in tissues that are not diseased. For example, in the treatment of cancer, it is necessary to use a high enough dose of a drug to kill abnormally proliferating cells without killing an unacceptably high number of normal cells. Thus, one of the major challenges of disease treatment is to identify ways of using cellular drug delivery vehicles to incorporate and to selectively release agents at a desired target site.
It has been suggested that red blood cells (RBCs) may be exploited as active agent/drug delivery vehicles (DeLoach and Sprandel 1985, Bibliotheca Haematologica; Publ. Karger, Munich) since it is possible to incorporate agents into human RBCs using a variety of loading techniques. An example of a loading technique is electroporation. During electroporation, red blood cell membranes are made transiently permeable by exposing the membranes to short pulses of high electric fields, thereby allowing agents of interest to enter the cells. The electroporation process allows high loading indices to be achieved within a very short time period (Flynn et al., 1994, Cancer Letts., 82, 225-229).
Loading of cells after osmotic shock followed by a recovery period to allow cells to recover isotonicity, and loading after hypotonic shock, followed by reverse hypotonic dialysis has also been performed (see, e.g., Luque and Pinilla, 1993, Ind Farmac. 8, 53-59).
The present invention provides a system for rendering a cell, such as a red blood cell, suitable for use as a delivery vehicle for delivering an agent (e.g., a drug) to a vertebrate.
The invention provides a loading system for loading a cell with an agent and for sensitising a cell to an energy field, thereby allowing a cell to release its contents, including the agent, in response to the energy field.
In one embodiment, the system comprises: a loading module for loading a cell with an agent; and a sensitisation module in fluid communication with the loading module, the sensitisation module for sensitising a cell to an energy field, such that the cell is induced to release the agent upon exposure to the energy field, and wherein the sensitisation module and the loading module are separate.
In one embodiment, the loading module comprises a mechanism for loading the cell by hypotonic dialysis.
In another embodiment, the loading module comprises one or more hollow fibers.
In a further embodiment, the system comprises a pre-sensitisation module for exposing a cell to a condition which increases the ability of a cell to be loaded in the loading module at least two-fold compared to a cell which is not pre-sensitised. In one embodiment, the presensitisation module and the sensitisation module are integral. In another embodiment, the presensitisation module and the sensitisation module are separate and are in fluid connection with each other.
In one embodiment, one or both of the sensitisation module and the pre-sensitisation module are in communication with a source of electrical energy.
In another embodiment, the sensitisation module comprises a chamber for receiving at least one cell, one or more walls of the chamber being defined by electrodes to enable an electric field to be established within the chamber. In one embodiment, at least one electrode has a crenellated or sinusoidal cross sectional profile. In another embodiment, the sensitisation module comprises one or more flow-through cuvettes. In still another embodiment, the sensitisation module comprises one or more micropores. In a further embodiment, the micropore comprise electrodes positioned to define a space capable of allowing passage of a cell. In one embodiment, the cell is a red blood cell. In another embodiment, the electrodes are tubular.
In one embodiment, the system further comprises a resealing module for resealing the cell subsequent to loading. In another embodiment, the system further comprises a monitoring module comprising a sensor for sensing the amount of agent which is loaded into the red blood cell. In a further embodiment, the system comprises a feedback mechanism adapted to receive a signal from the monitoring module and to alter one or more loading parameters to adjust the amount of agent loaded into the cell.
As described above, the system comprises a plurality of modules. The modules can be configured and/or operated in a number of different ways:
In a first embodiment of the invention, the system comprises a sensitisation module (S) and a loading module (L). The sensitisation module and loading module are in fluid connection with each other. The sensitisation module acts on cells to sensitize the cells such that the cells undergo lysis upon the subsequent application of an energy field such as ultrasound. The loading module enables the cells to be loaded with an agent of interest. The sensitisation module may be placed before or after the loading module, such that the cells are sensitised and subsequently loaded, or loaded and subsequently sensitised. In a preferred embodiment, the cells which are loaded are red blood cells.
In a second embodiment of the invention, the system comprises a pre-sensitisation module (P), a sensitisation module (S) and a loading module (L). The modules are in fluid connection with each other. The sensitisation and loading modules act on the cells, as described above and therefore may be connected to each other in any order. The pre-sensitisation module enables the cells to be pre-sensitised so that the cells will subsequently undergo efficient loading, and must therefore be placed before the loading module. The modules may therefore be connected in the following order: S, P, L; P, S, L; and P, L, S.
In a third embodiment of the invention, the system comprises a pre-sensitisation module and a loading module in fluid connection with each other. As noted above, the pre-sensitisation module acts on cells so that the cells will subsequently undergo efficient loading, and thus, this module is placed before the loading module. In a preferred embodiment, the cells are red blood cells.
In a fourth embodiment, the system comprises a pre-sensitisation/sensitisation module (referred to here as a xe2x80x9cbifunctional modulexe2x80x9d) and a loading module in fluid connection with each other. In this embodiment a single module is used to enable pre-sensitisation and sensitisation of cells. A number of ways of configuring the modules are available in this embodiment. In one option, the cells pass into the bifunctional module first for pre-sensitisation and then into the loading module for loading. After passing through the loading module, the cells pass back into the bifunctional module for a second time, where sensitisation of the cells takes place. In a preferred embodiment, the cells are red blood cells.
A further option is to pass the cells into the bifunctional module for pre-sensitisation, then back through the bifunctional module for a second time for sensitisation. After exiting the bifunctional module for a second time the cells are then fed into the loading module for loading. The bifunctional module therefore acts to pre-sensitise the cells on the first pass and sensitise the cell on the second pass. The reverse configuration may also be used, in which the bifunctional module sensitises the cells the first time, and pre-sensitises the cells the second time. The cells are then loaded with agent in the loading module. In a preferred embodiment, the cells are red blood cells.
The loading systems described above may include additional modules such as an optional washing module, and/or an optional resealing module. Thus, one or both of the washing and resealing modules may be included in any of the configurations described above. These may be placed after the loading modules. Further resealing and/or washing modules may also be placed after the pre-sensitisation and sensitisation modules. Monitoring modules may also be used in any of the above-described combinations and in any positions in the system.
The invention further provides a method for providing a cell suitable for delivery of an agent to a vertebrate, the method comprising the steps of: (a) providing an system according to the first embodiment of the invention; (b) loading the cell with an agent in the loading module of the system according to the first embodiment of the invention; and (c) sensitising the cell in the sensitising means of the system. In a preferred embodiment, the method is used to transform a red blood cell into a delivery vehicle for an agent.
In another embodiment, the method comprises the steps of: (a) providing a system according to the second embodiment of the invention; (b) loading the cell with an agent in the loading module of the system; and (c) pre-sensitising the cell in the pre-sensitising means of the system. In a preferred embodiment, the method is used to transform a red blood cell into a delivery vehicle for an agent.
In a further embodiment, the method comprises the use of an electroporation system for the sensitisation, or the pre-sensitisation of a cell, such as a red blood cell.